Abstract: TH-PO374

Uromodulin Is Essential for Correct Insertion of Uric Acid Transporter GLUT9 in the Plasma Membrane

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Koenigshausen, Eva, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Porwoll, Clara, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Probst, Paul, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Rump, Lars C., Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Sellin, Lorenz, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
Background

Uromodulin (UMOD) mutations cause autosomal dominant tubulointerstitial kidney disease (ADTKD). Patients with ADTKD-UMOD usually show hyperuricemia in childhood and progressive renal failure in the course of the disease. The pathogenesis of hyperuricemia by mutation in UMOD is scarcely understood so far. In microdissection analyses of tubular structures, UMOD has been localized also to the proximal tubulus and uric acid transporters have been localized to the distal proximal tubulus.

Methods

HEK293T cells expressing UMOD WT and uric acid transporters ABCG2, Urat1, OAT4, GLUT9, NPT1, NPT4 and UAT were lysed and UMOD was precipitated. On western blot, uric acid transporters were visualized. Subcellular fractionation experiments with UMOD WT or its mutant P236L and GLUT9 were performed. Cos7 cells were transfected with UMOD WT and P236L. Immunofluorescent labeling with markers for the endoplasmic reticulum (calnexin), Golgi (giantin), endosomes (EEA1) and the plasma membrane (WGA) were performed. In addition, a triple staining for UMOD WT or P236L, GLUT9 and organelle markers were done.

Results

UMOD WT interacts with the uric acid transporters ABCG2, URAT1, OAT4 and GLUT9. In the subcellular fractionation experiment GLUT9 is localized to the membrane fraction if UMOD WT is expressed. However, if UMOD P236L is expressed, GLUT9 localizes to the vesicular fraction. UMOD WT colocalizes with WGA (plasma membrane), while UMOD P236L colocalizes in the ER (calnexin). UMOD WT and GLUT9 colocalize at the plasma membrane and UMOD P236L localizes with GLUT9 in the ER.

Conclusion

UMOD WT interacts with several uric acid transporters and is essential for the proper insertion of uric acid transporters in the plasma membrane. With UMOD mutation P236L, mutated UMOD and uric acid transporters are trapped in the ER. This retention of uric acid transporters could explain the reduced fractional uric acid excretion with resulting hyperuricemia in patients with ADTKD-UMOD.